EP0677533A2 - Antikörper gegen Epitopen mit Homologie zu Auto-Antigenen, Verfahren zur Herstellung und dessen Verwendungen - Google Patents
Antikörper gegen Epitopen mit Homologie zu Auto-Antigenen, Verfahren zur Herstellung und dessen Verwendungen Download PDFInfo
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- EP0677533A2 EP0677533A2 EP95302440A EP95302440A EP0677533A2 EP 0677533 A2 EP0677533 A2 EP 0677533A2 EP 95302440 A EP95302440 A EP 95302440A EP 95302440 A EP95302440 A EP 95302440A EP 0677533 A2 EP0677533 A2 EP 0677533A2
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- C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
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- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
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- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/42—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins
- C07K16/4283—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an allotypic or isotypic determinant on Ig
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56966—Animal cells
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01K2217/00—Genetically modified animals
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Definitions
- This invention relates to methods of obtaining autologous monoclonal antibodies (AMABs) to self-antigens or homologs thereof, and the use of these antibodies in the analysis of cell populations and in cell separation techniques.
- AMABs autologous monoclonal antibodies
- Antibodies have proven useful in medical applications for both diagnosis and therapy, and in biotechnology applications including cell separation. More generally, their high degree of binding specificity facilitates their use in the identification and localization of any compound to which antibodies can be generated in conjunction with techniques as varied as electron microscopy and enzyme linked immunosorbent assays.
- Antibodies are comprised of both heavy and light chain polypeptides joined by interchain disulfide bonds and other intramolecular interactions. An individual heavy chain is paired with an individual light chain by these disulfide bonds. Of the different classes or isotypes of antibodies, three isotypes (IgD, IgE, and IgG) are comprised of two identical heavy chain/light chain pairs joined by a disulfide bond, and the remaining two isotypes (IgA and IgM) are comprised of more complicated polymers of identical heavy chain/light chain pairs. Each chain contains a constant region and a variable region. The constant regions are peculiar to the animal that generates the antibody and the specific isotype of antibody, while the variable regions conform to the structure of the epitope to which the antibody binds.
- antigen is used herein to refer to a substance, whether an entire molecule or a domain within a molecule, which is capable of eliciting production of antibodies with binding specificity to that substance. Further, the term antigen is applied herein to substances, which in wild type host organisms would not elicit antibody production by virtue of self-recognition, but can elicit such a response in a host animal with the appropriate genetic engineering.
- epitope is used herein to refer to the discrete, three-dimensional sites on an antigen, which are recognized by B lymphocytes. Epitopes are the immunologically active regions on a complex antigen, the regions that actually bind to a B-cell receptor, and that are actually bound by the resulting antibody molecules that are produced by the B-cell. Antigens generally contain at least one epitope and usually more than one epitope. Epitopes on protein antigens can be linear or non-linear. Linear epitopes are those comprised of contiguous amino acid residues in the amino acid sequence of a protein.
- Linear epitopes may or may not require conformational folding to form the native three-dimensional structure and elicit an immune response that produces antibodies with binding specificity to the antigen.
- Non-linear epitopes are comprised of non-contiguous amino acid residues. Thus, non-linear epitopes always require some degree of protein folding to bring the requisite amino acid residues into proximity of one another to form the native three-dimensional structure and elicit an immune response that produces antibodies with binding specificity to the antigen.
- the term "self” is used herein to describe antigens or epitopes which would not be recognized or be only poorly recognized by the B-cell receptors of a wild type member of the host species, by virtue of being included among the substances which are normally biosynthesized by the host species, or to which the host species is normally exposed. Such substances induce tolerance of the host immune system and the host is said to be “tolerized” to the substances.
- the vertebrate immune system is able to discriminate between self-antigens and foreign antigens, mounting an antibody-mediated immune response to the latter and not the former.
- the antibody response is mediated by the B cells.
- Variable-region gene rearrangements occur in an ordered sequence during B-cell maturation in the bone marrow.
- each B cell contains a single, functional variable-region DNA sequence encoding an immunoglobulin heavy chain and a single, functional variable-region DNA sequence encoding an immunoglobulin light chain. This process leads to the generation of mature, immunocompetent B cells each of which is antigenically committed to a single epitope.
- immunologic tolerance to "self” components is accomplished by the selective ablation of B cells with variable regions that are antigenically committed to self-epitopes.
- This self-tolerance precludes production of antibodies specific for antigens or epitopes that are synthesized by a host vertebrate. Thus, only antigens that contain epitopes which are recognized as foreign by the host can be used to generate antibodies.
- mice In mice, allogeneic differences between strains have allowed the production of mouse anti-mouse antibodies specific for proteins of which such allogeneic differences have been produced. Kessler et al. (1979) J. Immunol. 123:2772-2778; Reif and Allen (1964) J. Exp. Med. 120:413-433; Marshak-Rothstein (1979) J. Immunol. 122:2491-2497; and Oi and Herzenberg (1979) Molec. Immunol. 16:1005-1017. Initially, polyclonal antisera and then monoclonal antibodies specific for T cell surface proteins and mouse IgD antibodies were obtained in this manner. These antibodies, however, only recognize the gene product of particular mouse strains.
- FACS fluorescence activated cell sorting
- markers include antibodies, antibodies, and other methods reliant upon antibody affinity to particular cell surface proteins known as "markers”.
- FACS fluorescence activated cell sorting
- Such approaches to cell analysis and separation are especially useful for the determination of cell lineages, the isolation of cells which are capable of synthesizing a particular product, and the treatment of various disease conditions with specific cell types.
- highly purified hematopoietic stem cells are essential for hematopoietic engraftment including, but not limited to, cancer patients and transplantation of other organs in association with hematopoietic engraftment.
- Isolated cell populations are also important targets for gene therapy in the treatment of genetic disorders, AIDS and various forms of cancer.
- efficient purification of cells of such low concentration in the body requires antibodies which recognize and bind to stem cell specific markers with high specificity. Such antibodies are difficult to obtain due to the homology between human and murine stem cell markers.
- This invention seeks to provide novel methods of obtaining (autologous) monoclonal antibodies (AMABs) to a self-antigen or homolog thereof.
- the method can comprise obtaining a genetically engineered host animal that does not biosynthesize or synthesizes an altered form of at least one epitope within the self-antigen and utilizing the host lack of self-tolerance to the at least one epitope to produce antibodies specific to that antigen.
- the invention also encompasses the antibodies or any functional derivative thereof produced by the method.
- the invention further encompasses methods of isolating cells comprising utilizing the antibodies obtained by the method of the invention which are specific to a cell surface antigen.
- the invention seeks to provide novel methods of obtaining AMABs which have binding specificity to self-antigens or homologs thereof.
- the invention may provide a means for overcoming the limitation on producing AMABs to antigens that the host recognizes as self, as well as a method for obtaining "targeted antibodies” that is, AMABs with binding specificity to known, particular and extremely precise epitopes.
- the invention also allows for obtaining AMABs to biological molecules, or epitopes thereof, considered essential for growth and development. As discussed more fully herein, targeted genetic replacement allows for production of functional equivalents of the molecules and thus allows sufficient growth and development of the animal to produce antibodies.
- the term "homolog” is used herein to refer to an antigen with a structure that is so similar to an antigen produced by the host animal, a "self-antigen", as to preclude or seriously hinder the production of antibodies to the homolog.
- the phrase "self-antigen or homolog thereof” is used herein to signify that the invention is not directed solely to obtaining AMABs with binding specificity to self-antigens, but rather the invention is directed to obtaining AMABs to any compound with such a high degree of structural similarity to a self-antigen, that the homolog is sufficiently recognized as self that the host animal does not produce an adequate antibody-mediated immune response.
- An adequate antibody-mediated immune response is one in which either AMABs are not obtained or, if produced, they do not have high affinity for the antigen.
- target antigen is used herein to refer to the composition to which the host animal is exposed and against which an immune response is generated.
- the "target antigen epitope” is a region on the target antigen to which the AMABs bind.
- the term "immune response" is used herein to refer to the production of AMABs by B cells. These antibodies bind to a particular antigen regardless of whether they effect a change in the antigen such as providing immunity to a disease causing agent.
- the AMABs may be associated with the B cell surface and may also be freely circulating.
- structurally non-homologous is used herein as a description to compare self-antigens with antigens biosynthesized by host animals as a result of genetic engineering.
- Two antigens are structurally non-homologous, when an antibody can be generated that binds to one and not the other.
- Structurally non-homologous means that there is some structural difference, perhaps slight, between two or more antigens.
- the structurally non-homologous difference between two antigens can be as small as a single amino acid difference, or the presence or absence of a methyl group.
- the term "functionally equivalent” is used herein as a description to compare self-antigens and target antigens biosynthesized by host animals as a result of genetic engineering. In many cases, disrupting synthesis of a self-antigen can prove lethal, reduce survivorship or the overall health of genetically engineered host animals so as to interfere with obtaining antibodies. Thus, as described herein, genetic engineering can be used to eliminate the biosynthesis of a self-antigen and cause the production of another, functionally equivalent antigen. This functionally equivalent antigen exerts a sufficient amount of the self-antigen function to compensate for the deleterious elimination of the self-antigen by improving the rate of survivorship, overall health or immunocompetence of the host animal to the extent that AMABs may be obtained. As used herein, functionally equivalent antigens are understood to be structurally non-homologous.
- biosynthetic repertoire is used herein to refer to the sum total of all of the compounds biosynthesized by a given host animal.
- wild type is used herein to describe individuals of the host species and strain that have not been subjected to genetic engineering relating to the target antigen and are not descended from such a genetically engineered organism. Thus such individuals will usually produce the self-antigen of interest.
- genetically engineered is used to describe animals that have had one or more genes directly altered or eliminated so as to prevent or alter synthesis of a particular antigen or set of antigens. Such alteration or elimination is at the genetic level, for instance by specific genetic knockout and replacement. Genetic engineering of the target antigen is generally limited to the extent that antibodies can be obtained with enhanced binding specificity to at least one epitope on the antigen or set of antigens, as described herein. The term “genetically engineered” is also applied to the progeny of the originally altered animals.
- domain is used herein to refer to any region or portion of an antigen, including the whole antigen, any portion which is less than the whole antigen, or any region which is more than the whole antigen by virtue of the addition of components which may serve to mask at least one epitope.
- the host animal is genetically engineered so that it does not synthesize a particular self-antigen.
- the genetically modified host is immunized with the self-antigen or a homolog thereof, the host immune system does not recognize the antigen as self, and is thus able to produce an antibody-mediated immune response from which AMABs can be obtained.
- Methods of obtaining such "knock-out" mutants are known in the art and are described for instance, in Mansour et al. Nature 336:348-352 (1986).
- breeding may be used to achieve a homozygous mutant or to obtain multiply genetically engineered host animals.
- the genetic modification to the host can ultimately be acquired through germ-line transmission.
- Progeny of the genetically engineered mice are also encompassed by the invention. Any other method known in the art of creating transgenic animals is suitable for use herein. Suitable methods include, but are not limited to, those described by Kitamura et al. (1991) Nature 350 :423-426; Shinkai et al.(1993) Science 259 :822-825; and Komori et al. (1993) Science 261 :1171-1175. Briefly, in the case of self-antigens of the immune system, embryonic stem (ES) cells homozygous for the gene modification can be implanted into blastocysts of immunodeficient mice such as RAG-deficient mice. The reconstituted animal will, in many cases, lack the immune deficient phenotype and contain the gene modification in its lymphocytes.
- ES embryonic stem
- the host animal is genetically engineered such that the synthesis of a particular self-antigen is replaced by the synthesis of a functionally equivalent antigen.
- antibodies can be obtained for antigens the elimination of which would prove lethal, drastically reduce survivorship, or otherwise hamper efforts to obtain antibodies.
- the self-antigen that is eliminated from the biosynthetic repertoire of the host can be any compound, domain, or epitope thereof, that is normally synthesized by a wild type member of the host species.
- the antibodies obtained by the invention can be directed to any antigen, including but not limited to, proteins, carbohydrates, lipids, nucleic acids, enzyme cofactors or any naturally-occurring aggregates or covalently-linked combinations thereof, or any phosphorylated or sulfonated species thereof.
- the host can be genetically engineered in any way such that the biosynthesis of the antigen is eliminated or altered.
- knocking out the expression of a particular enzyme that is involved in the biosynthesis of the antigen can result in nonproduction of the antigen or the production of a functionally equivalent antigen.
- enzymes include, but are not limited to, enzymes involved in the polymerization and attachment of carbohydrates, phosphate groups, lipids, sulfur-containing groups to proteins could be eliminated, resulting in the elimination or change in structure of the compounds that are covalently attached to proteins, as in the synthesis of glycoproteins.
- antibodies with binding specificity to particular self-glycoproteins or carbohydrate structures on glycoproteins can be obtained by the methods of the invention.
- More global methods of eliminating or changing production of antigens may be utilized. These methods include, but are not limited to, eliminating synthesis of particular genes or gene families and eliminating particular cell types. Examples of suitable genes and gene families include those regulated by the presence of particular factors such as steroids or cytokines or genes which are expressed in a cell type specific manner. An example of such a cell type is brown fat cells.
- the antigen is a protein.
- a knockout mutation for a protein could involve preventing the synthesis of the entire protein (by removing the entire gene or by altering transcriptional or translational control elements) or eliminating the synthesis of just one antigenic domain, while allowing the remaining portion of the protein to be synthesized normally.
- the replacement of the self-antigen with a functionally equivalent antigen can be achieved by at least three distinct types of replacements although any formal replacement known in the art is suitable for use herein.
- the gene encoding the self-antigen can be replaced by a recombinant gene derived from the gene encoding the self-antigen.
- the gene encoding the protein is altered such that an antigenic region is deleted, replaced by an alternative amino acid sequence, or masked by the addition of a novel amino acid sequence.
- the genetically engineered elimination of the antigen can be as minor as the addition, elimination or substitution of a single amino acid.
- such small changes in the structure of the self-protein antigen allow AMABs to extremely precise epitopes to be obtained without severely disrupting the function of the self-antigen.
- a single amino acid change in a suitable domain would result in AMABs to epitopes that include the single amino acid.
- the gene encoding the self-antigen can be replaced by the gene that encodes a homologous protein obtained from an organism that is related to the host species, such that the encoded protein is functionally equivalent but at least partially antigenically non-equivalent. Closely related species usually have a high degree of sequence homology for the same protein, typically greater than 90%.
- This strategy is ideal for practicing the invention in cases in which expression of the antigen is critical to the health of the organism, and AMABs with binding specificity to a small number of epitopes is desired.
- This strategy can also be practiced by replacing only a region of the gene encoding the self-antigen with the corresponding region of the gene encoding the same antigen from a related species.
- the gene encoding the self-antigen can be replaced by a gene encoding a protein that is known to have the same or a similar function, but is structurally non-homologous. This strategy is particularly useful where expression of the antigen is critical to the health of the organism and AMABs with binding specificity to particular epitopes is not required.
- protein antigens to which the AMABs obtained by the invention can have binding specificity include, but are not limited to, cell surface antigens, for example adhesion molecules, MHC class I and class II molecules, integrin, cytokines, selectins, cytokine receptors and immunoglobulins.
- cell surface antigens for example adhesion molecules, MHC class I and class II molecules, integrin, cytokines, selectins, cytokine receptors and immunoglobulins.
- the invention can be practiced using any non-human vertebrate host animal, including fish, reptiles, amphibians, birds, and mammals.
- the host is preferably a mammal and usually belongs to an order including, but not limited to, rodentia , lagomorpha , primates , carnivora , perissodactyla and artiodactyla .
- the host is rodentia or murine and most preferably a mouse.
- AMABs are well known in the art and so are not described in detail herein. Any method known in the art of monoclonal antibody production can be used herein. Such methods include, but are not limited to, separating B cells with cell-surface antibodies of the desired specificity, cloning the DNA expressing the variable regions of the light and heavy chains and expressing the recombinant genes in a suitable host cell. Standard monoclonal antibody generation techniques can be used wherein the AMABs are obtained from immortalized antibody-producing hybridoma cells. These hybridomas can be produced by fusing B lymphocytes, preferably isolated from the immunized host spleen, with compatible immortalized cells, preferably a B cell myeloma.
- compositions of matter comprising the AMABs obtained by the methods described herein.
- the terms "AMAB(s)," “antibody” or “antibodies” include the entire antibody as well as antibody fragments containing functional portions thereof.
- the term “AMABs” includes any monospecific compound comprised of a sufficient portion of the light chain variable region and/or the heavy chain variable region to effect binding to the epitope to which the whole antibody has binding specificity.
- the fragments may include the variable region of at least one heavy or light chain immunoglobulin polypeptide, and include, but are not limited to, Fab fragments, Fab2 fragments, and Fv fragments.
- the monospecific domains may be attached by any method known in the art to another suitable molecule.
- the attachment may be, for instance, chemical or by genetic engineering.
- the AMABs may be produced by any recombinant means known in the art. Such recombinant AMABs include, but are not limited to, fragments produced in bacteria and AMABs in which the majority of the constant regions have been replaced by human antibody constant regions.
- such "humanized" AMABs may be obtained by breeding the genetically engineered host vertebrates described herein with a compatible transgenic animal that expresses functional human Ig loci in place of the wild type Ig loci.
- transgenic animals expressing human Ig loci see WIPO publication number WO 91/10741 and Rajewsky et al. DE P4228162.8. With successive crosses, host animals that do not express a particular self-antigen, but do express humanized Ig proteins can be obtained. When such animals are immunized they will produce humanized or partly humanized AMABs to particular self-antigens. Such humanized AMABs are preferred for use in therapeutic indications.
- the AMABs can be conjugated to other compounds including, but not limited to, enzymes, magnetic beads, colloidal magnetic beads, haptens, fluorochromes, metal compounds, radioactive compounds, drugs or haptens.
- the enzymes that can be conjugated to the AMABs include, but are not limited to, alkaline phosphatase, peroxidase, urease and ⁇ -galactosidase.
- the fluorochromes that can be conjugated to the AMABs include, but are not limited to, fluorescein isothiocyanate, tetramethylrhodamine isothiocyanate, phycoerythrin, allophycocyanins and Texas Red.
- the metal compounds that can be conjugated to the AMABs include, but are not limited to, ferritin, colloidal gold, and particularly, colloidal superparamagnetic beads.
- the haptens that can be conjugated to the AMABs include, but are not limited to, biotin, digoxygenin, oxazalone, and nitrophenol.
- radioactive compounds that can be conjugated or incorporated into the AMABs are known to the art, and include but are not limited to technetium 99m (99TC), 125I and amino acids comprising any radionuclides, including, but not limited to, 14C, 3H and 35S.
- 99TC technetium 99m
- 125I 125I
- amino acids comprising any radionuclides, including, but not limited to, 14C, 3H and 35S.
- Any drug known in the art that can be conjugated to protein by any method known in the art is suitable for use in the present invention.
- Such drugs can be conjugated to the AMABs for highly specific delivery to the target molecule.
- the invention further provides methods of using the AMABs, including but not limited to, immunoassays and separating cells.
- Suitable immunoassays are well known in the art and so need not be described in detail herein. These include, but are not limited to, ELISAs and RIAs.
- Methods of cell separation include, but are not limited to, separation based on secretion of molecules and separation based on cell surface molecules. Methods of separating cells based on secretion of molecules are described in Serial No. 07/965,934 and International Application No. PCT/US93/10126.
- Methods for separating cells based on specific cell surface markers generally include the steps of obtaining the AMABs by the methods described herein, contacting the AMABs to a heterogeneous population of cells, and performing a cell separation technique based on the affinity of the AMABs for the cell surface antigen. Any method known in the art may be employed to separate or isolate the cells by initially removing cells with particular cell surface antigens or of particular lineages.
- AMABs are particularly useful for identifying markers associated with particular cell lineages and/or stages of differentiation.
- the AMABs may be attached directly or indirectly to a solid support to allow for separation.
- the separation techniques employed should maximize the retention of viability of the fraction to be collected.
- Various techniques of different efficacy may be employed to obtain separations. Such separations are where up to 10%, usually not more than about 5%, preferably not more than about 1%, of the total cells present not having the marker may remain with the cell population to be retained.
- the particular technique employed will depend upon efficiency of separation, cytotoxicity of the methodology, ease and speed of performance, and necessity for sophisticated equipment and/or technical skill.
- Procedures for cell separation include, but are not limited to, magnetic separation using antibodies linked to colloidal magnetic particles, affinity chromatography and cytotoxic agents joined to a monoclonal antibody or used in conjunction with any antibody-dependent separation techniques known in the art.
- cells may be separated by "panning" with antibody attached to a solid matrix, e.g., a plate.
- Fluorescence activated cell sorting FACS
- FACS Fluorescence activated cell sorting
- Any antibody-dependent separation technique known in the art may be used in conjunction with both positive and negative separation techniques that rely on the physical properties of the cell rather than antibody affinity, including, but not limited to, elutriation and density gradient centrifugation.
- autologous monoclonal antibodies can be directly coupled to magnetic polystyrene particles like Dynal M 450 or similar magnetic particles and used, e.g., for cell separation.
- antibodies can be biotinylated or conjugated with digoxigenin and used in conjunction with avidin or anti-digoxigenin coated affinity columns like SEPARATE LC (Cellpro).
- autologous monoclonal antibodies are used in conjunction with colloidal superparamagnetic microparticles having an organic coating by, e.g., polysaccharides. Miltenyi et al.
- Cytometry 11:231-238 These particles can be used having a size of 10 to 200 nm, preferably between 40 and 100 nm, and can be either directly conjugated to autologous antibodies or used in combination with anti-immunoglobulin, avidin, or anti-hapten-specific microbeads.
- Polysaccharide-coated superparamagnetic particles are commercially available from Miltenyi Biotec GmbH, Germany.
- One procedure which may be used is first incubating the cells for a short period of time at reduced temperatures, generally about 4°C, with saturating levels of AMABs specific for a particular cell surface antigen, and then washing the cells with PBS and a fetal calf serum (FCS) cushion.
- the cells may then be suspended in a buffer medium as described above and separated on the basis of the AMABs for the particular determinants, using various protein(s) specific for the AMABs or AMAB-antigen complex.
- the AMABs may be conjugated with markers, including, but not limited to, magnetic beads, which allow for direct separation, biotin, which can be removed with avidin or streptavidin bound to a support, digoxigenin detected by anti-digoxigenin antibodies and fluorochromes, which can be used with a FACS, or the like, to allow for ease of separation of the particular cell type. Any technique may be employed which is not unduly detrimental to the viability of the remaining cells.
- the cells may then be separated by a FACS or other methodology known to the art.
- Multi-color analyses may be employed, by a variety of the methods including, but not limited to, FACS and fluorescence microscopy.
- the cells may be separated on the basis of the level of staining for the particular antigens.
- the invention further encompasses obtaining AMABs to self-antigens which are receptors by the method described herein and using such AMABs as pharmaceutical agents, wherein the AMABs demonstrate efficacy as receptor agonists or antagonists.
- mice The generation of IgD-deficient mice was performed as described by Roes and Rajewsky (1993) J. Exp. Med. 177:45-55. Briefly, the strategy of C ⁇ gene inactivation and the screening procedure for positive clones was as described in Example 1.
- Targeted ES cell clones were injected into blastocyst isolated from C57BL/6 mice and transferred to (C57BL/6 x BALB/c) fosters.
- Male chimeric offspring were mated with C57BL/6 females for germ-line transmission of the ⁇ T mutation.
- Offspring derived from ES cells were identified by coat color and analyzed for the presence of the mutation which was called ⁇ T, by Southern blotting or phenotypically, by flow cytometry.
- Homozygous mutant mice ( ⁇ T/ ⁇ T) were obtained by the interbreeding of heterozygous offspring.
- poly(A)+ RNA isolated from spleens of homozygous mutant ( ⁇ T/ ⁇ T) and wild-type mice was analyzed by Northern blotting.
- mRNA containing C ⁇ exons spliced to the C ⁇ transmembrane exon would be larger than the normal C ⁇ transcripts of 2.4 ( ⁇ s) or 2.7 kb ( ⁇ m), because the 3'-untranslated region of the ⁇ message is 600-bp longer than that of the ⁇ message.
- Hybridization of splenic poly(A)+ RNA of homozygous mutant mice with a C ⁇ transmembrane-specific probe reveals bands of 4.8-, 4.0-, 3.8-, and 3.0-kb. However, none of these bands hybridized with the C ⁇ -specific probe.
- the detection limit of the two probes was similar within a factor of two (1.2 x 106 copies for the C ⁇ and 0.6 x 106 for the ⁇ m probe) as judged by the signals obtained from hybridization to standard plasmid DNA.
- the C ⁇ probe is a cDNA fragment of 1 kb with complete match to the mRNA and the standard plasmid, whereas the ⁇ m probe hybridizes to the mRNA over a stretch of only 480 bp, but 700 bp on the plasmid standard because it contains the ⁇ m1/m2 intron. Consequently, a mRNA representing C ⁇ exons spliced to the ⁇ m exons, if detected with the ⁇ m probe, should also be revealed with the C ⁇ probe. Because the 3.0-, 3.8-, 4.0-, and 4.8-kb bands are clearly above the ⁇ m probe detection limit, they should, if containing C ⁇ sequences, also be detected with the C ⁇ probe. This, however, is not the case.
- mRNA species representing potentially functional chimeric ⁇ / ⁇ molecules were undetectable in Northern blots using as much as 10 ⁇ g of splenic poly(A)+ RNA of ⁇ T/ ⁇ T mice. Because mRNA encoding the ⁇ H chain can be detected with the C ⁇ m-specific probe in as little as 300 ng of poly(A)+ RNA of spleens of normal mice, a putative mRNA encoding the extracellular domains of ⁇ and the transmembrane portion of ⁇ would be at least 30-fold less abundant than ⁇ H chain message in normal mice, if it exists at all. Thus, mRNA potentially able to encode an IgD-like molecule is undetectable in homozygous mutant mice ( ⁇ T/ ⁇ T) by northern blot analysis.
- IgD-deficient mice were generated by gene-targeting as described in Examples 1 and 2. One animal obtained thereby was immunized intraperitoneally (i.p.) with mouse monoclonal antibody of class IgD (267.7 ⁇ "a" allotype) precipitated in alum.
- Hybridomas obtained were screened for production of anti-IgD antibodies by ELISA using plates coated with B1-8 (IgD), BSA (bovine serum albumin), 267.7 (IgD), R33-24-12 (anti-IgM) and developed using R33-18-10.1-Biotin (rat anti-mouse Kappa) and R33-60-Biotin (anti-IgM) antibodies.
- clones showed reactivity to B1-8 (IgD b ), thirteen of these showed reactivity to 267.7 (IgD a ), one of these was of class IgM. Two other clones showed reactivity to IgM but not to IgD. Hybridomas were further characterized for relative binding affinity and isotype. Fifteen of 21 clones tested showed IgG1 isotype. Additionally, clones were characterized for binding to trypsin-treated and untreated mouse spleen cells using biotinylated anti-mouse IgG1 and streptavidin-phycoerythrin. Trypsin cuts surface IgD and allows localization of the epitope on the IgD molecules.
- Spleen cells obtained from a B6 x 129 mouse were stained with the fluorescein-conjugated AMAB ⁇ 1.3 and phycoerythrin-conjugated anti-mouse IgM antibody (R33-24), washed and analyzed in a FACScan flow cytometer gating on lymphocytes and excluding dead cells by propidium iodide staining.
- the column was washed using one backflush procedure using a 23G flow resistor and the retained cells were eluted. All fractions were analyzed by flow cytometry (FACScan). Dead cells were identified by propidium iodide staining were excluded from analysis.
- Figure 4 shows the histograms of a FACS analysis of the separations.
- the first row depicts the cells before separation
- the second row depicts the unmagnetic fraction
- the third row depicts positive cells.
- ⁇ 1.3 results are in the left column
- ⁇ 3.5 results are in the right column.
- the data indicate that at least 95% of the IgD expressing cells are retained by the column, whereas the IgD cells being retained and eluted from the column have a purity of at least 92%. Background staining in this separation and analysis is mainly caused by macrophages taking up antibodies in an unspecific manner.
- NCAMs Neural cell adhesion molecules
- NCAMs are members of the immunoglobulin super family mediating homo- and heterophilic cell-cell interactions. NCAMs appear in various isoforms generated by alternative splicing. Hemperly et al. (1986) Proc. Natl. Acad. Sci. USA 83 :3037-3041; Barthels et al. (1987) EMBO J. 6 :907-914; and Barthels et al. (1992) Eur. J. Neurosci. 4 :327-337. During embryonic development, NCAMs are expressed in derivatives of all three germ layers whereas in the adult animal they are predominantly present in neural tissue.
- Heterozygous animals of two lines were mated and gave rise to 78 offspring. Of these, 38 animals (49%) were heterozygous, 22 (28%) were wild type and 18 (23%) were homozygous for the mutated allele, indicating an almost perfect Mendelian distribution. Homozygous mutant animals are fertile and appear healthy up to four months of age even though they are about 10% smaller by weight than wild type and heterozygous littermates. Isolated brains of a mutant and a heterozygous animal had obvious anatomical differences: the olfactory bulb was reduced in size in mutants compared to +/+ and +/- animals; and the brain weight was reduced by about 10% (after correction for body weight).
- mice described above are inoculated with an antigenic amount of an NCAM suspended in a suitable adjuvant.
- Booster shots are given as required and the antibody titer is measured periodically. Once the titer is sufficient, a suitable method for generation of AMABs is followed.
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Cited By (7)
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EP1606387A2 (de) * | 2003-03-04 | 2005-12-21 | Alexion Pharmaceuticals, Inc. | Zur erzeugung konstanter hybridbereiche verwendete vektoren |
US7026456B1 (en) * | 1998-01-23 | 2006-04-11 | Hoffman-La Roche, Inc. | Antibodies against human IL-12 |
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US8084020B2 (en) * | 2002-05-01 | 2011-12-27 | Beth Israel Deaconess Medical Center | Use of anti-CD1 antibodies for the modulation of immune responses |
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US7119248B1 (en) * | 1994-04-12 | 2006-10-10 | Miltenyi Biotec Gmbh | Antibodies against epitopes with homology to self antigens, methods of preparation and applications thereof |
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US7026456B1 (en) * | 1998-01-23 | 2006-04-11 | Hoffman-La Roche, Inc. | Antibodies against human IL-12 |
US8716449B2 (en) | 1998-01-23 | 2014-05-06 | Hoffman-Laroche, Inc. | Antibodies against human IL-12 |
WO2001098357A3 (en) * | 2000-06-19 | 2003-01-03 | Beth Israel Hospital | Compositions and methods of monoclonal and polyclonal antibodies specific for t cell subpopulations |
EP2336187A3 (de) * | 2000-06-19 | 2012-01-11 | Beth Israel Deaconess Medical Center | Zusammensetzungen und Verwendungen von monoklonalen oder polyklonalen Antikörpern, die spezifisch für T-Zell Subpopulationen sind |
US8138314B2 (en) | 2000-06-19 | 2012-03-20 | Beth Israel Deaconess Medical Center, Inc. | Compositions and methods of monoclonal and polyclonal antibodies specific for T cell subpopulations |
US8084020B2 (en) * | 2002-05-01 | 2011-12-27 | Beth Israel Deaconess Medical Center | Use of anti-CD1 antibodies for the modulation of immune responses |
EP1606387A2 (de) * | 2003-03-04 | 2005-12-21 | Alexion Pharmaceuticals, Inc. | Zur erzeugung konstanter hybridbereiche verwendete vektoren |
EP1606387A4 (de) * | 2003-03-04 | 2008-04-23 | Alexion Pharma Inc | Zur erzeugung konstanter hybridbereiche verwendete vektoren |
WO2007080404A2 (en) * | 2006-01-12 | 2007-07-19 | Asterion Limited | Leptin ligand |
WO2007080404A3 (en) * | 2006-01-12 | 2008-03-06 | Asterion Ltd | Leptin ligand |
US9932402B2 (en) | 2011-10-27 | 2018-04-03 | Nkt Therapeutics Inc. | Humanized antibodies to iNKT |
CN109475109A (zh) * | 2016-05-20 | 2019-03-15 | 瑞泽恩制药公司 | 用于使用多个引导rna来破坏免疫耐受性的方法 |
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JP2006151991A (ja) | 2006-06-15 |
JP4362121B2 (ja) | 2009-11-11 |
JPH0856692A (ja) | 1996-03-05 |
EP0677533B1 (de) | 2005-01-12 |
JP3797680B2 (ja) | 2006-07-19 |
ATE286913T1 (de) | 2005-01-15 |
CA2146693C (en) | 2010-09-14 |
CA2146693A1 (en) | 1995-10-13 |
US7119248B1 (en) | 2006-10-10 |
JP2006061158A (ja) | 2006-03-09 |
DK0677533T3 (da) | 2005-05-30 |
EP0677533A3 (de) | 2000-04-12 |
ES2236698T3 (es) | 2005-07-16 |
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US20070101446A1 (en) | 2007-05-03 |
DE69533912T2 (de) | 2005-12-08 |
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